Since the confocal core facility opened in 1998, usage has steadily and rapidly increased from 194 orders (1,261 samples) in 1998 to 1,384 orders (10,836 samples) in FY 2008. Over 130 users from 25 different CCR laboratories or branches used the facility this year. Fifty two of these were new users that required extensive confocal training and supervision. Among the techniques now in use within our CCR confocal core facility are methods to visualize live cells in three dimensions over time, including the tracking of cell motion in three-dimensional tissue, in vivo imaging of differentiated embryonic stem cells in 3D, spontaneous fusion and asymmetric division of cancer stem cells, monitoring membrane fluidity of MDR cells using FRAP, and time lapse imaging of live cells expressing GFP-PKC isoforms to determine mode of drug action. Examples of projects using 2-photon technology include whole live lung imaging with 2-photon to determine metastatic potential of GFP expressing tumor cells as well as a very interesting project where an in vitro model of Osteosarcoma recreated the tumor microenvironment in tissue culture using nanofibers to mimic the collagen framework and allowed examination of the interaction between stromal cells and pre-metastatic cells. Another exciting area of expertise includes methods of in vivo biochemistry in which light microscopy is being used to assess whether two proteins physically interact at a certain time and place within a cell using Fluorescence Resonance Energy Transfer (FRET) technology as well as the use of FRET to measure drug potencies within the context of cell type. In addition, we have multi-plexed Quantum Dots (QDs) on lymphatic tissue and were able to image simultaneously five different QDs using the META detector technology which allowed us to custom design the emission filters needed. Our work on a clinical project involving the imaging and quantitation of the recruitment of DNA repair enzymes with the NCTVL involved not only training on the 2-photon confocal, but also the development of an imaging software macro in ImageProPlus to facilitate the analysis. This required training the investigators in the use of this imaging software and macro. A recent project uses second harmonic generation and autofluorescence with the 2-photon to image live lung sections and evaluate structural/morphological differences after tumor cell expansion. Another collaborative project investigated the DNA damage response, specifically the serine 81 phosphorylation of TDP1, a DNA repair enzyme, that promotes binding to XRCC1 and enhances the mobilization of TDP1 to the sites of damage together with XRCC1. Using FRAP analysis with the confocal, the recruitment kinetics of RFP-tagged TDP1 wild type versus TDP1 mutant (S81A) demonstrated that phosphorylation of serine 81 enhances the recruitment of TDP1 to the DNA damage site. The confocal was used for colocalization analysis of CARP-2, a negative regulator of TNF-induced NF-kB activation, and endosomes. Results from these studies indicated that CARP-2 is important in the endocytic vesicles where it limits the intensity of TNF-induced NF-kB activation by the elimination of one of the signaling components within the receptor complex.